Bioprocess for the high-yield production of food flavor-acceptable jasmonic acid and methyl jasmonate

ABSTRACT

Described is a bioprocess for the high-yield production of food flavor-acceptable jasmonic acid and methyl jasmonate, as well as a novel jasmonic acid isomer produced thereby and organoleptic uses thereof. The process yields at least 5% of the &#34;cis&#34; isomer defined according the structure:(wherein R is hydrogen or methyl) or at least 5% of the &#34;cis&#34; isomer defined according to the structure:(wherein R is hydrogen or methyl). Compositions containing at least 98% of the isomer having the structure:with an optical rotation (alphaD20) of +58° are novel. Compositions containing at least 98% of the isomer having the structure:with an optical rotation (alphaD20) of +58° are also novel. The process of our invention comprises the cultivation under aerobic condition of one or more specific strain of Diplodia gossypina in a nutrient medium followed either by (1) isolation of the jasmonic acid product or (2) esterification of the jasmonic acid to form methyl jasmonate followed by the isolation of the methyl jasmonate and novel products produced by such process.

BACKGROUND OF THE INVENTION

This invention concerns a microbial process for production ofcompositions of matter containing isomers of jasmonic acid having thestructure:

which are flavor acceptable as well as isomers having the structure:

which are also flavor acceptable. This invention is also concerned withthe production of methyl jasmonate isomers from the aforementionedjasmonic acid isomers.

Considerable time and effort have been expended by microbiologists inthe search for better processes for the production of jasmonic acid andmethyl jasmonate which are flavor acceptable isomers, including thoseisomers defined according to the structure:

wherein R is methyl or hydrogen. Thus, Broadbent in United KingdomPatent Specification No. 1,286,266 published on Aug. 23, 1972 disclosesand claims a process for the manufacture of jasmonic acid whichcomprises cultivation of the organism Lasiodiplodia theobromae in anutrient medium containing an assimilable source of carbon and anassimilable source of nitrogen followed by isolation of the product fromthe culture medium. Günther, et al, German Democratic Republic Patent DD279 688 published on Jun. 13, 1990 discloses a process for theproduction of 7-iso-jasmonic acid by strains of the organismBotryodiplodia theobromae in aerobic culture. This work is alsodiscussed in the paper Miersch, et al, Phytochemistry, Volume 26, No. 4,pages 1037-1039, 1987, entitled “(+)-7-ISO-JASMONIC ACID AND RELATEDCOMPOUNDS FROM BOTRYODIPLODIA THEOBROAME.” The organisms, Botryodiplodiatheobromae and Lasiodermea theobromae are synonyms of Diplodia gossypinaas discussed in the paper Jones, MYCOTAXON, Volume VI, No. 1 at pages24-26, July-September 1997 (title: “THE CURRENT TAXONOMIC STATUS OFDIPLODIA GOSSYPINA.” As confirmed by Häusler and Münch in the articleentitled “Microbial Production of Natural Flavors,” Volume 63, No. 10,ASM News at pages 551-559:

“Another plant fatty acid metabolite, jasmonic acid, an endogenous plantgrowth regulator with a variety of physiological functions, is producedby means of a similar metabolic pathway. After a lipoxygenase produces ahydroperoxide derivative of linolenic acid, this compound is convertedto its allene oxide, which cyclizes. β-Oxidation and double-bondreduction yields jasmonic acid. The methylester of jasmonic acid is notonly a volatile plant hormone, possibly involved in interplantcommunication, but is also an important flavor and fragrance moleculethat imparts a sweet-floral, jasmine-like note . . .

Otto Miersch and his collaborators at the Institute of PlantBiochemistry, Halle-Saale, Germany, who were studying fungal plantpathogens, including Botryodiplodia theobromae, discovered that suchmicroorganisms produce jasmonic acid. The biosynthetic steps leading tojasmonic acid in this filamentous fungus are probably similar to thosefound in plants. Recently our laboratory, which is evaluating thisstrain's capacity for producing jasmonic acid, found that B. theobromaeyields only very low concentrations of jasmonic acid in liquid culture.Such findings suggest that the biosynthesis and excretion of jasmonicacid is strictly controlled during the growth cycle of this fungus onplants in its natural habitat.”no bioprocesses for the high yieldproduction of fruit flavor-acceptable jasmonic acid or methyl jasmonateexist. The Häusler and Münch paper was published in October 1997. ASMNews is published by the American Society for Microbiology.

Thus, in the flavor and fragrance art, a need has arisen for thedevelopment and efficient high yield production of naturally occurringjasmonic acid and methyl jasmonate, which have heretofore been found tobe useful and necessary for the creation of flavor formulations used inaugmenting or enhancing the aroma or taste of foodstuffs, chewing gums,toothpaste, medicinal products, chewing tobaccos and smoking tobaccosand also useful in augmenting or enhancing the aroma of perfumecompositions, colognes and perfumed articles (e.g., solid or liquidanionic, cationic, nonionic or zwitterionic detergents, perfumedpolymers, fabric softener compositions, fabric softener articles, hairpreparations, cosmetic powders and the like).

Methyl jasmonate (without indicating which isomer) is disclosed byArctander, Perfume and Flavor Chemicals at monograph 2093 to have a“Powerful floral-herbaceous, sweet-tenacious odor representing typicalbackground notes of Jasmin absolute.” Arctander further discloses thatthis material “is an almost obvious candidate for work on improvedartificial Jasmin absolute.” Acree, et al, J. Agric. Food Chem., 1985,Volume 33, pages 425-427, discloses that the isomer of methyl jasmonatehaving the structure:

and having an optical rotation (α_(D) ²⁰) of +58 has a strong odor,whereas the other stereoisomers of (Z)-methyl jasmonate aresubstantially odorless.

No specific disclosures exist in the prior art showing the use of thejasmonic acid isomers of our invention defined according to thestructure:

or defined according to the structure:

for use as flavor adjuvants (wherein R is methyl or hydrogen).Furthermore, nothing in the prior art discloses the stereoisomer of thecompound having the structure:

particularly the stereoisomer having the optical rotation (α_(D) ²⁰) of+58°.

THE INVENTION

Our invention relates to a bioprocess for the high yield production offood flavor acceptable jasmonic acid and methyl jasmonate, a noveljasmonic acid isomer produced thereby and uses thereof. The process ofour invention yields at least 5% of the “cis” isomer defined accordingto the structure:

(wherein R is hydrogen or methyl) or the “cis” isomer defined accordingto the structure:

(wherein R is hydrogen or methyl). Compositions containing at least 98%of the isomer having the structure:

having an α_(D) ²⁰ of +58° are novel. Furthermore, compounds having thestructure:

wherein R is methyl or hydrogen having an α_(D) ²⁰ of +58° are alsonovel.

The process of our invention comprises the cultivation under aerobicconditions of one or more specific strains of Diplodia gossypina in anutrient medium followed by either (1) isolation of the jasmonic acid or(2) esterification of the jasmonic acid to form methyl jasmonatefollowed by isolation of the methyl jasmonate. More specifically, ourprocess comprises cultivation under aerobic conditions of a strain ofDiplodia gossypina organism selected from the group consisting of:

(i) Diplodia gossypina ATCC 10936;

(ii) Diplodia gossypina ATCC 20575;

(iii) Diplodia gossypina NRRL 25011; and

(iv) Diplodia gossypina ATCC 36037

in a nutrient medium containing an assimilable source of carbon and anassimilable source of nitrogen followed by isolation of the jasmonicacid product from the culture medium or followed by esterification ofthe jasmonic acid to form methyl jasmonate and then followed byisolation of the methyl jasmonate product from the reaction mass.

In one embodiment of the process of our invention, the compound,10-oxo-trans-8-decenoic acid having the structure:

is intimately admixed in the nutrient medium in an amount of from about0.7 up to about 10 ppm (parts per million) by weight of the nutrientmedium prior to the cultivation step.

The 10-oxo-trans-8-decenoic acid having the structure:

is disclosed in U.S. Pat. No. 5,681,738 issued on Oct. 28, 1997 to be afungal growth hormone to stimulate mycelial growth of cultivatedmushrooms. The specification of U.S. Pat. No. 5,681,738 issued on Oct.28, 1997 is incorporated by reference herein. The bioprocess of ourinvention is shown according to the reactions:

wherein D.G. strain is intended to mean “strain of Diplodia gossypina.”

The esterification reaction following the bioprocess reaction of ourinvention is shown as follows:

Thus, the assimilable carbon source for our invention has been found tobe glucose. The preferred assimilable nitrogen source is sodium nitrate.

Preferably, the aeration rate range for carrying out the cultivationstep of our invention is from about 0.5 v/v/m (liters air per literfermentation mass per minute) up to about 1.0 v/v/m.

Furthermore, it is preferable that the culture medium contain magnesiumion, for example, magnesium ion in the form of magnesium sulfateheptahydrate having the formula:

MgSO₄.7H₂O

with the magnesium ion concentration ([Mg⁺⁺]) being in the range of fromabout 6×10⁻³ up to about 10.5×−1 gram moles per liter. Thus, forexample, the concentration range of magnesium sulfate heptahydrate ingrams per liter preferably is in the range of from about 1.5 up to about2.5 grams per liter of magnesium sulfate heptahydrate.

The cultivation step of the process of our invention may take placeunder liquid surface culture conditions or may take place undersubmerged liquid culture conditions. The incubation time is from about 3up to about 15 days.

The pH range during the fermentation is from about 4.5 up to about 9,with a preferred pH of about 6.

The temperature for the fermentation reaction may vary between about 20°C. up to about 35° C., with a preferred temperature range for thefermentation being from about 25° C. up to about 32° C.

We have found that the preferred strain of Diplodia gossypina is theDiplodia gossypina ATCC 10936.

Along with the desired “cis” jasmonic acid isomers having thestructures:

prepared according to the reactions:

other jasmonic acid isomers defined according to the structures:

for example, are also produced in very small quantities when using thestrains of Diplodia gossypina:

(i) Diplodia gossypina ATCC 10936;

(ii) Diplodia gossypina ATCC 20575;

(iii) Diplodia gossypina NRRL 25011; and

(iv) Diplodia gossypina ATCC 36037.

The inventive process may be conducted in a batch or continuous mode ofoperation. In a batch fermentation, the nutrient broth, culture andsubstrate, are combined and fermented until the jasmonic acidconcentration becomes constant. In a continuous process, the substratein the nutrient broth may be continuously recirculated through afermentation reactor, with the provision that substrate and product arerespectively added and removed from the recirculating broth.

In carrying out the present invention, cultivation and fermentiveincubation of the Diplodia gossypina fungus are accomplished in anaqueous medium in the presence of the usual nutrient substances(preferably using sodium nitrate, magnesium sulfate heptahydrate andglucose). A suitable medium thus is one which contains carbon sources,nitrogen sources, inorganic salts and growth factors. Additionalexamples of inorganic salts include the phosphate salts of magnesium andthe phosphate and sulfate salts of sodium, calcium and potassium. Thesenutrients may be supplemented with, for example, one or more vitamins ofthe “B” group and one or more trace minerals such as iron, manganese,cobalt and copper, as desired.

For the nutrient broth, it is preferred to utilize glucose (as stated,supra) at a concentration of from about 2 up to about 20 weight percent,preferably at about 10 weight percent. It is also preferred to employ“B” vitamins either as a separate supplement or in the form of a yeastextract. The kind and amounts of these additives can be determined byapplying the general knowledge in the art for the cultivation ofmicroorganisms.

In a typical procedure, one of the Diplodia gossypina organisms:

(i) Diplodia gossypina ATCC 10936;

(ii) Diplodia gossypina ATCC 20575;

(iii) Diplodia gossypina NRRL 25011; or

(iv) Diplodia gossypina ATCC 36037

is first cultivated in inoculum quantities to produce a mature culturein nutrient broth. The culture is inoculated into a fermentor nutrientbroth and allowed to establish itself. The substrate is then added andfermentation is continued until a steady concentration of jasmonic acidis present.

The cultivation and fermentative incubation of the Diplodia gossypinafungus can be carried out as a stationary culture or as a submergedculture (e.g., shake-flask fermentor) under aerobic conditions.Cultivation and incubation may proceed as stated, supra, in a pH rangeof from about 4.5 up to about 9, preferably at 6. The pH may beregulated by the addition of an inorganic or organic acid or base suchas hydrochloric acid, acetic acid, sodium hydroxide, calcium carbonate,ammonia, ion-exchange resins or by the addition of a buffer such asphosphate or phthalate. The incubation temperature as stated, supra, issuitably maintained between about 20° C. up to about 35° C., with arange of from about 25° C. up to about 32° C. being preferred.

In accordance with another typical procedure of the present invention,the process is conveniently carried out by adding the substrate to theculture medium at the onset of cultivation under aerobic conditions.Alternatively, the substrate may be added either alone or in combinationwith another carbon source, such as xylose during fermentativeincubation or when cultivation is complete. It is preferable to add thesubstrate to the culture medium during the period of from 4 up to 24hours after the growth of the culture in the fermentative broth hascommenced. Desirable results can be obtained when the substrate is addedcontinuously over the entire fermentation after an initial fungal(Diplodia gossypina) cultivation period of from 3 up to 12 hours. Apreferred feed rate for this continuous addition is from about 0.01 upto 1 gram per hour per liter, with a preferred range of from 0.6 up to0.8 grams per hour per liter. The concentration of the substrate in themedium may vary depending on the conditions employed. In practice, theconcentration of the substrate in the medium may conveniently vary from0.01% up to about 10%, with a preferable concentration of about 1% byweight consistent with the manner in which it is added to the culture.

The present invention provides unexpectedly high yields of jasmonicacid, e.g., from about 0.8 up to about 1.5 grams per liter.

Another aspect of our invention is the production of methyl jasmonateisomers having the structure:

and having the structure:

In this case, the isolation step comprises the steps of:

(a) extraction of the jasmonic acid from the fermentation broth with anextraction solvent such as ethyl acetate to form ajasmonic acid extract;

(b) concentration of the jasmonic acid extract whereby the extractionsolvent is stripped;

(c) esterification of the resulting jasmonic acid with methyl alcoholwhereby the methyl jasmonate is formed according to one of thereactions:

(d) concentration of the resulting methyl jasmonate.

Additional steps of fractionation of the resulting concentrate with, forexample, silica gel, in order to effect isomer separation and thencollecting the 98% “cis” isomer are preferred.

Thejasmonic acid derivative(s) defined according to the structure:

or according to the structure:

(wherein R is methyl or hydrogen) and one or more auxiliary perfumeingredients including, for example, hydrocarbons, alcohols, ketones(other than the jasmonic acid derivatives of our invention), aldehydes,nitrites, esters (other than the jasmonic acid derivatives of ourinvention), ethers, synthetic essential oils and natural essential oils,may be admixed so that the combined odors of the individual componentsproduce a pleasant and desired fragrance, particularly and preferably inthe floral area (e.g., jasmine and jasmine/rose aromas). Such perfiumecompositions usually contain (a) the main note or the “bouquet” orfoundation stone of the composition; (b) modifiers which round off andaccompany the main note; (c) fixatives which include odorous substanceswhich lend a particular note to the perfume throughout all stages ofevaporation, and substances which retard evaporation; and (d) topnoteswhich are usually low-boiling, fresh-smelling materials.

In perfume compositions, it is the individual components whichcontribute to their particular olfactory characteristics; however, theoverall sensory effect of the perfume composition will be at least thesum total of the effects of each of the ingredients. Thus, one or moreof the jasmonic acid derivative(s) of our invention can be used toalter, modify or enhance the aroma characteristics of a perfumecomposition, for example, by utilizing or moderating the olfactoryreaction contributed by another ingredient in the composition. Thus, asuitable mixture would be that of the cis isomers of jasmonic acid andmethyl jasmonate, to wit: the mixture of the compounds having thestructures:

or the mixture of compounds having the structures:

The amount of jasmonic acid derivative(s) of our invention, which willbe effective in perfume compositions as well as in perfumed articles andcolognes, depends upon many factors including the other ingredients,their amounts and the side effects which are desired. It has been foundthat perfume compositions containing as little as 0.005% of the jasmonicacid derivative(s) or even less (e.g., 0.002%) can be used to impartpowerfuL long lasting jasmine, floral-herbaceous aromas withsweet-herbaceous, green-woody topnotes to soaps, cosmetics, detergentsincluding anionic, cationic, nonionic and zwitterionic solid or liquiddetergents, perfumed polymers and other products. The amounts employedcan range up to 70% of the fragrance components and will depend uponconsiderations of cost, nature of the end product, the effect desired onthe finished product and the particular fragrance sought.

The jasmonic acid derivative(s) of our invention are useful (taken aloneor taken together with other ingredients in perfume compositions) indetergents, soaps, space odorants and deodorants, perfumes, colognes,toilet waters, bath preparations, hair preparations such as lacquers,brilliantines, pomades and shampoos; cosmetic preparations such ascreams, deodorants, hand lotions and sun screens; powders such as talcs,dusting powders, face powders and the like.

As little as 0.25% of the jasmonic acid derivative(s) will suffice toimpart an intense and long lasting jasmine, floral-herbaceous aroma withsweet-herbaceous, green-woody topnotes to floral perfume formulations.Generally no more than 5% of the jasmonic acid derivative(s) based onthe ultimate end product are required to be used in the perfumecompositions.

Furthermore, as little as 0.25% of the jasmonic acid derivative(s) willsuffice to impart such aromas to perfumed articles per se, whether inthe presence of other perfume materials or whether used by themselves.Thus, the range of use of the jasmonic acid derivative(s) of ourinvention in perfumed articles, e.g., perfumed polymers and solid orliquid anionic, cationic, nonionic or zwitterionic solid or liquiddetergents, may vary from 0.25% up to about 5% by weight based on thetotal weight of the perfiuned article.

In addition, the perfume composition or fragrance composition of ourinvention can contain a vehicle or carrier for the jasmonic acidderivative(s). The vehicle can be a liquid such as a nontoxic alcohol,e.g., ethanol; a nontoxic glycol e.g., propylene glycol; or the like.The carrier can also be an absorbent solid such as a gum (e.g., gumarabic, xanthan gum or guar gum) or components for encapsulating thecomposition by means of coacervation (such as gelatin) or by means offormation of a polymer around a liquid center (as by using a ureaformaldehyde prepolymer to form a polymeric capsule around a perfumecomposition center).

It will be appreciated from the present disclosure that the jasmonicacid derivative(s) according to the present invention can be used toalter, vary, fortify, modify, enhance or otherwise improve the flavor ofa wide variety of materials which are ingested, consumed or otherwiseorganoleptically sensed.

The terms “alter” and “modify” in their various forms will be understoodherein to mean the supplying or imparting of a flavor character or noteto an otherwise bland, relatively tasteless substance, or augmenting anexisting flavor characteristic where the natural flavor is deficient insome regard or supplementing the existing flavor impression to modifyits organoletpic character.

The term “enhance” is intended herein to mean the intensification (byuse of the jasmonic acid derivative of our invention) of a flavor oraroma note or nuance in a tobacco flavor or foodstuff or perfumecomposition or perfumed article without changing the quality of saidnote or nuance.

A “flavoring composition” is taken to mean one which contributes a partof the overall flavor impression by supplementing or forfing a naturalor artificial flavor in a material, or one which supplies substantiallyall the flavor and/or aroma character to a consumable article.

The term “foodstuff” as used herein includes both solid and liquidingestible materials for man or animals, which materials usually do, butneed not, have nutritional value. Thus, foodstuffs include meats,gravies, soups, convenience foods, malt, alcoholic and other beverages,milk and dairy products, seafoods, including fish, crustaceans, mollusksand the like, candies, vegetables, cereals, soft drinks, snacks, dog andcat foods, other veterinary products and the like. The jasmonic acidderivative(s) of our invention are also useful in tobacco flavorants andtobacco enhancers.

The term “tobacco” will be understood herein to mean natural productssuch as, for example, Burley, Turkish tobacco, Maryland tobacco,flue-cured tobacco and the like, including tobacco-like or tobacco-basedproducts such as reconstituted or homogenized leaf and the like, as wellas tobacco substitutes intended to replace natural tobacco such aslettuce and cabbage leaves and the like. The tobaccos and tobaccoproducts in which the jasmonic acid derivative(s) of our invention areuseful include those designed or used for smoking such as in cigarettes,cigar and pipe tobacco, as well as products such as snuff, chewingtobacco and the like.

When the jasmonic acid derivative(s) of this invention are used in aflavoring composition, they can be combined with conventional flavoringmaterials or adjuvants. Such co-ingredients or flavor adjuvants are wellknown in the art for such use and have been extensively described in theliterature. Requirements of such adjuvant materials are: (1) that theybe non-reactive with the jasmonic acid derivative(s) of our invention;(2) that they be organoleptically compatible with the jasmonic acidderivative(s) of our invention whereby the flavor of the ultimateconsumable material to which the jasmonic acid derivative(s) are addedis not detrimentally affected by the use of the adjuvant; and (3) thatthey be ingestibly acceptable and thus nontoxic or otherwisenon-deleterious. Apart from these requirements, conventional materialscan be used and broadly include other flavor materials, vehicles,stabilizers, thickeners, surface active agents, conditioners and flavorintensifiers.

Such conventional flavoring materials include saturated fatty acids,unsaturated fatty acids and amino acids; alcohols including primary andsecondary alcohols, esters, carbonyl compounds including ketones (otherthan the jasmonic acid derivatives of our invention) and aldehydes;lactones; other cyclic organic materials including benzene derivatives,alicyclic compounds, heterocyclics such as furans, pyridines, pyrazinesand the like; sulfur-containing compounds including thiols, sulfides,disulfides and the like; proteins; lipids, carbohydrates; so-calledflavor potentiators such as monosodium glutamate; magnesium glutamate,calcium glutamate, guanylates and inosinates; natural flavoringmaterials such as cocoa, vanilla and caramel; essential oils andextracts such as anise oil, clove oil and the like and artificialflavoring materials such as vanillin, ethyl vanillin and the like.

Specific preferred flavor adjuvants are as follows:

anise oil;

ethyl-2-methyl butyrate;

vanillin;

cis-3-heptenol;

cis-3-hexenol;

trans-2-heptenal;

butyl valerate;

2,3-diethyl pyrazine;

methyl cyclopentenolone;

benzaldehyde;

valerian oil;

3,4-dimethoxyphenol;

amyl acetate;

amyl cinnamate;

γ-butyryl lactone;

furfural;

trimethyl pyrazine;

phenyl acetic acid;

isovaleraldehyde;

ethyl maltol;

ethyl vanilin;

ethyl valerate;

ethyl butyrate;

cocoa extract;

coffee extract;

peppermint oil;

spearmint oil;

clove oil;

anethol;

cardamom oil;

wintergreen oil;

cinnamic aldehyde;

ethyl-2-methyl valerate;

γ-hexenyl lactone;

2,4-decadienal;

2,4-heptadienal;

methyl thiazole alcohol (4-methyl-5-β-hydroxyehtyl thiazole);

2-methyl butanethiol;

4-mercapto-2-butanone;

3-mercapto-2-pentanone;

1-mercapto-2-propane;

benzaldehyde;

furfural;

furfuryl alcohol;

2-mercapto propionic acid;

alkyl pyrazine;

methyl pyrazine;

2-ethyl-3-methyl pyrazine;

tetramethyl pyrazine;

polysulfides;

dipropyl disulfide;

methyl benzyl disulfide;

alkyl thiophene;

2,3-dimethyl thiophene;

5-methyl furfural;

acetyl furan;

2,4-decadienal;

guiacol;

phenyl acetaldehyde;

β-decalactone;

d-limonene;

acetoin;

amyl acetate;

maltol;

ethyl butyrate;

levulinic acid;

piperonal;

ethyl acetate;

n-octanal;

n-pentanal;

n-hexanal;

diacetyl;

monosodium gulatamate;

monopotassium glutamate;

sulfur-containing amino acids, e.g., cysteine;

hydrolyzed vegetable protein;

2-methylfuran-3-thiol;

2-methyldihydrofuran-3-thiol;

2,5-dimethylfuran-3-thiol;

2 hydrolyzed fish protein;

tetramethyl pyrazine;

propylpropenyl disulfide;

propylpropenyl trisulfide;

diallyl disulfide;

diallyl trisulfide;

dipropenyl disulfide;

dipropenyl trisulfide;

4-methyl-2-[(methylthio)-ethyl]-1,3-dithiolane;

4,5-dimetyl-2-(methylthiomethyl)-1,3-dithiolne; and

4-methyl-2-(methylthiomethyl)-1,3-dithiolane.

The jasmonic acid derivative(s) of our invention or compositionsincorporating them, as mentioned above, can be combined with one or morevehicles or carriers for adding them to the particular product. Vehiclescan be edible or otherwise suitable materials such as ethyl alcoholpropylene glycol, water and the like, as described supra. Carriersinclude materials such as gum arabic, carrageenan, xanthan gum, guar gumand the like.

The jasmonic acid derivative(s) prepared according to our invention canbe incorporated with the carriers by conventional means such asspray-drying, drum-drying and the like. Such carriers can also includematerials for coacervating the jasmonic acid derivative(s) of ourinvention to provide encapsulated products, as set forth supra. When thecarrier is an emulsion, the flavoring composition can also containemulsifiers such as mono- and diglycerides or fatty acids and the like.With these carriers or vehicles, the desired physical form of thecompositions can be prepared.

The quantity of jasmonic acid derivative(s) utilized should besufficient to impart the desired flavor characteristic to the product,but on the other hand, the use of an excessive amount of the jasmonicacid derivative(s) is not only wasteful and uneconomical, but in someinstances, too large a quantity may unbalance the flavor or otherorganoleptic properties of the product consumed. The quantity used willvary depending upon the ultimate foodstuff; the amount and type offlavor initially present in the foodstuff; the further process ortreatment steps to which the foodstuff will be subjected; regional andother preference factors; the type of storage, if any, to which theproduct will be subjected; and the preconsumption treatment such asbaking, frying and so on, given to the product by the ultimate consumer.Accordingly, the terminology “effective amount” and “sufficient amount”is understood in the context of the present invention to bequantitatively adequate to alter the flavor of the foodstuff.

It is accordingly preferred that the ultimate composition contain fromabout 0.1 parts per million (ppm) up to about 500 ppm of the jasmonicacid derivative(s).

The jasmonic acid derivative(s) of our invention when utilized inflavoring compositions can be varied over a wide range depending uponthe particular flavor nuances desired to be added to the foodstuff.Thus, the amounts of jasmonic acid derivative(s) of our invention may becontained in flavoring materials from about 1 ppm up to about 50% byweight of the flavoring composition. Indeed, the compounds having thestructures:

may be utilized in raspberry flavors (e.g., for use inraspberry-flavored yogurt, for example) at levels of between about 1%and about 50%. Such materials also have utility in flavorings for saladdressings, particularly in such cuisines as “Thai” cuisine.

According to another aspect of our invention, an organolepticallyimproved smoking tobacco product and additives therefor as well asmethods of making the same which overcome specific problems heretoforeencountered in which specific Turkish, oriental-like aromas prior tosmoking and improved Turkish, oriental aromas on smoking in themainstream and the side stream are created or enhanced or modified oraugmented and may be readily controlled and maintained at the desireduniform level regardless of variations in the tobacco components of theblend. In particular, low grade Virginia-type tobaccos may be upgradedusing the jasmonic acid derivative(s) of our invention.

This invention further provides improved tobacco additives and methodswhereby various desirable natural aromatic Turkish tobacco flavoringcharacteristics with oriental notes may be imparted to smoking tobaccoproducts and may be readily varied and controlled to produce the desireduniform flavoring characteristics.

In carrying out this aspect of our invention, we add to smoking tobaccomaterials or a suitable substitute therefor (e.g., dried lettuce leaves)an aroma and flavor additive containing as an active ingredient one ormore of the jasmonic acid derivative(s) of our invention.

In addition to the jasmonic acid derivative(s) of our invention, otherflavoring and aroma additives may be added to the smoking tobaccomaterial or substitute therefor, either separately or in admixture withthe jasmonic acid derivative(s) of our invention as follows:

I. Synthetic Materials

β-Ethyl-cinnamaldehyde;

Eugenol;

Dipentene;

β-Damascenone;

Maltol;

Ethyl maltol;

Delta-Undecalactone;

Delta-Decalactone;

Benzaldehyde;

Amyl acetate;

Ethyl butyrate;

Ethyl valerate;

Ethyl acetate;

2-Hexenol-1;

2-Methyl-5-isopropyl-1,3-nonadiene-8-one;

2,6-Dimethyl-1,6-undecadiene-10-one;

2-ethyl-5-isopropyl acetophenone;

2-Hydroxy-2,5,5,8a-tetramethyl-1-(2-hydroxyethyl)-decahydronnaphthalene;

Dodcahydro-3a,6,6,9a-tetramethyl naphthol(2,1-b) furan;

4-Hydroxy hexanoic acid, γ-lactone; and

Polyisoprenoid hydrocarbons defined in Example V of U.S. Pat. No.3,589,372 issued on Jun. 29, 1971.

II. Natural Oils

Celery seed oil;

Coffee extract;

Bergamot oil;

Cocoa extract;

Nutmeg oil; and

Origanum oil.

An aroma and flavoring concentrate containing one or more of thejasmonic acid derivative(s) of our invention and, if desired, one ormore of the above-indicated additional flavoring additives may be addedto the smoking tobacco material, to the filter or to the leaf or paperwrapper. The smoldng tobacco material may be shredded, cured, cased andblended tobacco material or reconstituted tobacco material or tobaccosubstitutes (e.g., lettuce leaves) or mixtures thereof. The proportionsof flavoring additives may be varied in accordance with taste, butinsofar as enhancement or the imparting of oriental and/or Turkishtobacco notes, we have found that satisfactory results are obtained ifthe proportion by weight of the sum total of jasmonic acid derivative(s)to smoking tobacco material is between 50 ppm and 1,500 ppm(0.005%-0.15%) of the active ingredients to the smoking tobaccomaterial. We have further found that satisfactory results are obtainedif the proportion by weight of the sum total of jasmonic acidderivative(s) used to flavoring material is between 500 and 15,000 ppm(0.05%-1.5%).

Any convenient method for incorporating the jasmonic acid derivative(s)into the tobacco product may be employed. Thus, the jasmonic acidderivative(s) taken alone or along with other flavoring additives may bedissolved in a suitable solvent such as ethanol, diethyl ether and/orother organic solvents, and the resulting solution may either be spreadonto the cured, cased and blended tobacco material or the tobaccomaterial may be dipped into such solution. Under certain circumstances,a solution of the jasmonic acid derivative(s) taken alone or takenfurther together with flavoring additives as set forth above may beapplied by means of a suitable applicator such as a brush or roller onthe paper or leaf wrapper for the smoking product, or it may be appliedto the filter by either spraying or dipping or coating.

Furthermore, it will be apparent that only a portion of the tobacco orsubstitute therefor need be treated, and the thus-treated tobacco may beblended with other tobaccos before the ultimate tobacco product isformed. In such cases, the tobacco treated may have the jasmonic acidderivative(s) in excess of the amounts or concentrations above indicatedso that when blended with other tobaccos, the final product will havethe percentage within the indicated range.

In accordance with one specific example of our invention, an aged, curedand shredded domestic Virginia tobacco is sprayed with a 20% alcoholsolution of the compound having the structure:

optical rotation (α_(D) ²⁰) equal +58° on a dry basis. Thereafter, thealcohol is removed by evaporation and the tobacco is manufactured intocigarettes by the usual techniques. The cigarette, when treated asindicated, has a desired and pleasing aroma which is detectable in themainstream and the side stream when the cigarette is smoked. The aromais described as being sweeter with pronounced Turkish/orientalcharacteristics and with improved body and enhanced tobacco characterwith subsidiary “floral,” “jasmonic” nuances in the mainstream and theside stream. In addition, interesting amber nuances are imparted.

While our invention is particularly useful in the manufacture of smokingtobacco such as cigarette tobacco, cigar tobacco and pipe tobacco, othertobacco products formed from sheeted tobacco dust or fines may also beused. Likewise, the jasmonic acid derivative(s) of our invention can beincorporated with materials such as filter tip materials, seam paste,packaging materials and the like, which are used along with tobacco toform a product adapted for smoking. Furthermore, the jasmonic acidderivative(s) can be added to certain tobacco substitutes of natural orsynthetic origin (e.g., dried lettuce leaves) and, accordingly, by theterm “tobacco” as used throughout this specification, is meant anycomposition intended for human consumption by smoking or otherwise whencomposed of tobacco plant parts or substitute material or both. Thus,chewing tobacco is also included in the foregoing meaning of “tobacco.”

During the fermentation reaction of our invention, two byproducts arealso formed, to wit, the compound having the structure:

and the compound having the structure:

the compound having the structure:

formed via the reaction:

It will be understood that each of the compounds having the structures:

may be retained with the jasmonic acid derivative(s) of our compositionfor their flavor or fragrance utilities. Thus, in addition to theadditives as set forth, supra, the compounds having the structures:

may be “retained” and not “separated” in the isolation of the jasmonicacid derivative(s) prior to the jasmonic acid derivative(s) beingutilized in flavor or fragrance formulations.

More specifically, our invention also contemplates mixtures ofderivatives having the structures:

(wherein R is methyl or hydrogen) taken further together with thecompounds having the structures:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the HPLC profile for the reaction product of Example XIII(conditions: 250 mm×4.6 mm ZORBAX® SB-C18 column).

FIG. 2 is the HPLC profile for the reaction product of Example XIV(conditions: 250 mm×4.6 mm ZORBAX® SB-C18 column).

FIG. 3 is the HPLC profile for the reaction products of Example XVhaving the structures:

FIG. 4 is the NMR spectrum for the product of Example XV for thecompound having the structure:

(optical rotation (α_(D) ²⁰)=+58).

FIG. 4A is an enlargement of section “A” of the NMR spectrum of FIG. 4.

FIG. 4B is an enlargement of section “B” of the NMR spectrum of FIG. 4.

FIG. 4C is an enlargement of section “C” of the NMR spectrum of FIG. 4.

FIG. 5A is a GC mass spectrum for the compound having the structure:

(α_(D) ²⁰=+58°) prepared according to Example XV.

FIG. 5B is the GC mass spectrum for the product produced according toExample XV having the structure:

together with small amounts of the compound defined according to thestructure:

with the optical rotation for the compound having the structure:

being (α_(D) ²⁰)+58°.

FIG. 6 is the mass spectrum for the compound having the structure:

prepared according to Example XV.

FIG. 7 is the NMR spectrum for the compound having the structure:

used in the process of Example XIII.

FIG. 8 represents a cutaway side elevation view of apparatus used informing perfumed polymers which contain embedded therein at least one ofthe jasmonic acid derivative-containing compositions of our invention.

FIG. 9 is a front view of the apparatus of FIG. 8 looking in thedirection of the arrows.

FIG. 10A is a schematic block flow diagram showing process steps forpreparing pure jasmonic acid defined according to the structures:

FIG. 10B is a schematic block flow diagram showing the process steps andapparatus for preparing the methyl jasmonate of our invention from thejasmonic acid of our invention according to the reactions:

FIG. 11 is the NMR spectrum for the pure optical isomer (α_(D) ²⁰) ofthe compound having the structure:

prepared according to Example XV having an optical rotation of +58°.

FIG. 11A is an enlargement of section “A” of the NMR spectrum of FIG.11.

FIG. 11B is an enlargement of section “B” of the NMR spectrum of FIG.11.

FIG. 11C is an enlargement of section “C” of the NMR spectrum of FIG.11.

FIG. 12 is the mass spectrum for the reaction product of Example XVcontaining the compounds having the structures:

as well as the compounds having the structures:

and the compound having the structure:

FIG. 13A is the HPLC profile for the optical isomer having thestructure:

prepared according to Example XV (optical rotation (α_(D) ²⁰)=+580)(conditions: OV1 column).

FIG. 13B is the HPLC profile for the optical isomer (α_(D) ²⁰=+58°)having the structure:

prepared according to Example XV (conditions: Carbowax column).

FIG. 14 is the HPLC profile (chiral column) for the optical isomer(α_(D) ²⁰=+58°) of the compound having the structure:

produced according to Example XV.

FIG. 15 is the NMR spectrum for the compound having the structure:

(optical isomer: (α_(D) ²⁰=+58°) prepared according to Example VI.

FIG. 15A is an enlargement of section “A” of the NMR spectrum of FIG.15.

DETAILED DESCRIPTION OF THE DRAWINGS

Referring to FIG. 1, the peak indicated by reference numeral 10 is thepeak for the jasmonic acid isomer defined according to the structure:

Referring to FIG. 2, the peak indicated by reference numeral 20 is thepeak for the jasmonic acid defined according to the structure:

Referring to FIG. 3, the peak indicated by reference numeral 41 is thepeak for the isomer having the structure:

The peak indicated by reference numeral 31 is for the isomer having thestructure:

The curve for the material containing the isomer having the structure:

is indicated by reference numeral 30. The curve for the materialcontaining the isomer shaving the structure:

is indicated by reference numeral 40.

Referring to FIG. 5B, the peak indicated by reference numeral 50 is thepeak for the isomer having the structure:

The peaks indicated by reference numerals 51 and 52 are for the isomerhaving the structure:

Referring to FIGS. 8 and 9, there is provided a process for formingscented polymer elements (wherein the polymer may be a thermoplasticpolymer such as low density polyethylene or polypropylene or copolymersof ethylene and vinyl acetate or mixtures of polymers and copolymerssuch as copolymers of ethylene and vinyl acetate and polyethylene) suchas pellets useful in the formation of plastic particles useful infabricating certain articles which may be perfumed. This processcomprises heating the polymer or mixture of polymers to the meltingpoint of said polymer or mixture of polymers, e.g., 250° C. in the caseof low density polyethylene. The lower most portion of the container ismaintained at a slightly lower temperature and the material in thecontainer is taken off at such location for delivery through theconduit. Thus referring to FIGS. 8 and 9, in particular, the apparatusused in producing such elements comprises a device for forming thepolymer containing perfume, e.g., polyethylene or polyethylene-polyvinylacetate of mixtures of same or polypropylene, which comprises a vat orcontainer 1212 into which the polymer taken alone or in admixture withother polymers in the perfuming substance which is at least one of thejasmonic acid derivative(s) of our invention or mixtures of jasmonicacid derivatives and other compatible perfume components is placed. Thecontainer is closed by means of an airtight lid 1228 and clamped to thecontainer by bolts 1265. A stirrer 1273 traverses the lid or cover 1228in an airtight manner and is rotatable in a suitable manner. Asurrounding cylinder 1212A having heating coils which are supplied withelectric current through cable 1214 from a rheostat or control 1216 isoperated to maintain the temperature inside the container 1212 such thatthe polymer in the container will be maintained in the molten or liquidstate. It has been found advantageous to employ polymers at such atemperature that the viscosity will be in the range of 90-100 Sayboltseconds. The heater 1218 is operated to maintain the upper portion ofthe container 1212 within a temperature range of, for example, 220-270°C. in the case of low density polyethylene. The bottom portion of thecontainer 1212 is heated by means of heating coils 1212A regulatedthrough the control 1220 connected thereto through a connecting wire1222 to maintain the lower portion of the container 1212 within atemperature range of 220-270° C.

Thus, the polymer or mixture of polymers added to the container 1212 isheated from 10-12 hours, whereafter the perfume composition or perfumematerial which contains one or more of the jasmonic acid derivatives ofour invention is quickly added to the melt. Generally, about 10-45% byweight of the resulting mixture of the perfumery substance is added tothe polymer.

After the perfume material is added to the container 1212. the mixtureis stirred for a few minutes, for example, 5-15 minutes, and maintainedwithin the temperature ranges indicated previously by the heating coil1212A. The controls 1216 and 1220 are connected through cables 1224 and1226 to a suitable supply of electric current for supplying the powerfor heating purposes.

Thereafter, the valve “V” is opened, permitting the mass to flowoutwardly through conduit 1232 having a multiplicity of orifices 1234adjacent to the lower side thereof. The outer end of the conduit 1232 isclosed so that the liquid polymer in intimate admixture with one or moreof the jasmonic acid derivatives of our invention or mixture of perfumesubstance and one or more of the jasmonic acid derivatives of ourinvention, will continuously drop through the orifices 1234 downwardlyfrom the conduit 1232. During this time, the temperature of the polymerintimately admixed with the perfumery substance in the container 1212 isaccurately controlled so that a temperature in the range of from about240-250° C. (for example, in the case of low density polyethylene) willexist in the conduit 1232. The regulation of the temperature through thecontrols 1216 and 1220 is essential in order to insure temperaturebalance to provide for the continuous dropping or dripping of moltenpolymer intimately admixed with the perfume substance which is all orwhich contains one or more of the jasmonic acid derivatives of ourinvention, through the orifices 1234 at a rate which will insure theformation of droplets 1236 which will fall downwardly onto a movingconveyor belt 1238 caused to run between conveyor wheels 1240 and 1242beneath the conduit 1232.

When the droplets 1236 fall onto the conveyor 1238, they form pellets1244 which harden almost instantaneously and fall off the end of theconveyor 1238 into a container 1245 which is advantageously filled withwater or some other suitable cooling liquid to insure the rapid coolingof each of the pellets 1244. The pellets 1244 are then collected fromthe container 1245 and utilized for the formation of other functionalproducts, e.g., garbage bags and the like.

Belt 1238 is continuously moistened with sponge-like material 1256supplied with water 1254 from container 1250 having sidewall 1248 sothat the belt is continuously cooled.

Referring to FIG. 10A, carbon source (e.g., glucose) from location 102is admixed with nutrient (containing, for example, magnesium sulfateheptahydrate and sodium nitrate) from location 101 and culture(containing at least one strain of Diplodia gossypina) from location 100are admixed and placed in fermenter 103. The fermenter is run, forexample, for a period of 10 days, and the resulting product is thenpassed through line 106 past valve 107 into mixing vessel 105 andcombined with extraction solvent (e.g., ethyl acetate) from location104. The resulting mixture is then separated and the solvent/jasmonicacid material is passed through line 110 past valve 111 intodistillation column 112 where overhead solvent is distilled through line113 and recovered at location 114, and crude jasmonic acid (bottoms) ispassed into vessel 119 where it is admixed with aqueous sodium carbonatesolution from location 116 which is passed through line 117 past valve118. The resulting sodium carbonate solution/jasmonic acid material isthen passed through line 121 past valve 122 and mixed with extractionsolvent from location 120 which is passed through line 123 past valve124 and admixed with such extraction solvent in vessel 125. The organicphase is then passed to location 127 through line 126, and the aqueousphase is passed through line 128 into vessel 131 where it is admixedwith phosphoric acid from location 129 (pH adjustment). The resultingproduct is then further mixed with extraction solvent from location 132which is passed through line 133 past valve 134 and admixed in vessel136. The resulting organic phase is separated from the aqueous phase andpassed through line 137 past valve 138 into distillation column 139where the recovered solvent (overhead) is passed through line 140 intosolvent recovery system 141, and the resulting pure jasmonic acid(bottoms) is passed through line 142 into location 143 (structure:

Referring to FIG. 10B, jasmonic acid having the structure:

(from the apparatus of FIG. 10A) at location 143 is passed through line145 past valve 146 into autoclave 149 where it is admixed with methanolfrom location 144 which is passed through line 147 past valve 148 intoautoclave 149. Autoclave 149 is heated under pressure using heatingelement 150 causing the reactions:

to take place thereat. The resulting products (crude) having thestructures:

are then passed through line 151 past valve 152 into distillation column153, where the excess methanol is recovered overhead via line 154 intorecovery system 155, and the resulting crude methyl jasmonate productsare admixed with extraction solvent from location 156, passed throughline 157 past valve 160 into vessel 159. The extraction solvent/crudemethyl jasmonate is then mixed and passed through line 161 past valve162 into distillation column 163, where solvent is recovered overheadvia line 164 into solvent recovery system 165, and purified methyljasmonate (bottoms) is passed into vessel 167, the purified methyljasmonate having the structures:

Referring to FIG. 12, the GC mass spectrum for the crude reactionproduct of Example XV contains the following peaks:

(a) the peak indicated by reference numeral 120 is the peak for thecompound having the structure:

(b) the peak indicated by reference 121 is the peak for the compoundhaving the structure:

(c) the peak indicated by reference numeral 121 also contains a sidepeak for the compound having the structure:

(d) the peaks indicated by reference numerals 122 and 123 are forcompounds having the structures:

(e) the peak indicated by reference numeral 124 is for the compoundhaving the structure:

(f) peaks indicated by reference numerals 125A and 125B are forcompounds defined according to the structure:

(g) the peak indicated by reference numeral 126 is for the compoundhaving the structure:

The following examples are given to illustrate embodiments of theinvention as it is preferred to practice it. It will be understood thatthese examples are illustrative, and the invention is not to beconsidered as restrictive thereto except as indicated in the appendedclaims.

All parts, proportions, percentages and ratios hereinafter referred toare by weight unless otherwise indicated.

EXAMPLE I Jasmonic Acid Screening Procedure

Medium 5059H KH₂PO₄ 1.0 gm MgSO₄.7H₂O 0.5 gm Yeast extract 1.0 gm Soypeptone 5.0 gm Trace minerals 10.0 ml Dextrose 30.0 gm Deionized water1.0 L pH adjusted to 5.5 before sterilization

Trace Minerals Solution 5059H FeSO₄.7H₂O 10.0 mg ZnSO₄.7H₂O 8.8 mgCuSO₄.5H₂O 15.0 mg MnSO₄.H₂O 7.6 mg (NH₄)₆Mo₇O₂₄.4H₂O 10.0 mg Deionizedwater 1.0 L

Procedure

500 Ml flasks containing 100 ml of broth were inoculated from slantstock cultures (PDA) and incubated at 25° C. stationary or at 150 rpm.Flask cultures were periodically analyzed by acidifying a 1 ml sample,extracting with 1 ml of ethyl acetate, centrifuging, concentrating andspotting on TLC plates. Alternately, a whole flask culture was xtractused to prepare the methyl ester and followed by GC analysis.

TLC Solvents Ethyl acetate 50% Hexanes 50% Acetic acid 0.5% 

TABLE I Jasmonic Acid (mg/L) Incubation Incubation Station- Time TimeMicroorganisms ATCC ary (Days) Shake (Days) Diplodia gossypina 10936 2107 1.1 7 Diplodia gossypina 16391 Neg 7 5.6 7 Diplodia gossypina 20575Neg 7 Neg 7 Diplodia gossypina 20576 Neg 7 Neg 7 Diplodia gossypina22644 Neg 7 Neg 7 Diplodia gossypina 26123  23 7 Neg 7

TABLE II Jasmonic Acid (TLC) Incu- Incu- Sta- bation bation tion- TimeTime Microorganisms ary (Days) Shake (Days) Diplodia gossypina ATCC Neg9 28570 Diplodia gossypina ATCC + 9 34643 Diplodia gossypina ATCC + 936037 Diplodia gossypina ATCC + 9 76087 Lasiodiplodia theobromae IFO + 531643 Botryosphaeria rhodina CBS Neg 7 110.11 Botryosphaeria rhodina CBSNeg 7 124.13 Botryosphaeria rhodina CBS Neg 7 174.26 Botryosphaeriarhodina CBS Neg 7 175.26 Botryosphaeria rhodina CBS Neg 7 176.26Botryosphaeria rhodina CBS Neg 10  190.73 Botryosphaeria rhodina CBS Neg7 230.30 Botryosphaeria rhodina CBS + 7 287.47 Botryosphaeria rhodinaCBS Neg 7 301.36 Botryosphaeria rhodina CBS + 5 304.79 Botryosphaeriarhodina CBS Neg 5 306.58 Botryosphaeria rhodina CBS Neg 5 356.59Botryosphaeria rhodina CBS Neg 10  374.54 Botryosphaeria rhodina CBS Neg10  456.78 Botryosphaeria rhodina CBS Neg 10  494.78 Botryosphaeriarhodina CBS Neg 10  495.78

Medium 5074H NaNO₃ 2.0 gm KH₂PO₄O 1.0 gm MgSO₄.7H₂O 0.5 gm KCl 0.5 gmFeSO₄.7H₂O 10.0 mg Yeast extract 1.0 gm Trace minerals 1.0 ml Dextrose50.0 gm Deionized water 1.0 L pH adjusted to 5.5 before sterilization

Trace Minerals Solution 5059H ZnSO₄.7H₂O 1.0 gm CuSO₄.5H₂O 0.15 gmMnSO₄.H₂O 0.1 gm (NH₄)₆Mo₇O₂₄.4H₂O 0.1 gm Deionized water 1.0 L

Screening Results

TABLE III Jasmonic Acid (TLC) Incu- Incu- Sta- bation bation tion- TimeTime Microorganisms ary (Days) Shake (Days) Botryodiplodia theobromaeFPRL Neg 7 Neg 7 S-22L Botryosphaeria rhodina CBS + 7 + 7 176.26Botryosphaeria rhodina CBS + 7 − 7 205.75 Botryosphaeria rhodina CBS +7 + 7 287.47 Botryosphaeria rhodina CBS + 7 + 7 304.79

TABLE IV NOTE: Same medium as 5074H, except pH adjusted to 9.0 beforesterilization. A 72-96 hour shake flask inoculum (100 ml/500 ml flask;150 rpm; 30° C.) was chopped in a sterile blender jar and 4 ml was usedto inoculate a new flask, including the addition of 10 ml sterile 50%dextrose. Jasmonic Acid (TLC) Incu- Incu- Sta- bation bation tion- TimeTime Microorganisms ary (Days) Shake (Days) Diplodia gossypina ATCC + 620575 Diplodia gossypina ATCC + 7 26123 Diplodia gossypina ATCC + 636037 Diplodia gossypina ATCC + 6 64959 Botryosphaeria rhodina CBS + 7287.47 Botryosphaeria rhodina CBS + 6 304.73 Lasiodiplodia theobromaeIFO + 7 6469

EXAMPLE II Screening of Microorganisms in Submerged Culture for theProduction of Jasmonic Acid

Medium: Buffered Minimal Salts Medium (bMS) NaNO₃ 2.0 gm/L KH₂PO₄ 2.0gm/L K₂HPO₄ 0.5 gm/L MgSO₄-7H₂O 2.0 gm/L KCl 0.5 gm/L FeSO₄-7H₂O 1.0mg/L TASTONE ® 900 0.5 gm/L

After sterilization of bMS medium at 120° C. and 15 psi, 60 g/l of 50%sterile glucose solution was added.

Procedure

Inocula for all microorganisms were prepared from frozen cultures. 2 Mlof inoculum were added to 100 ml of sterile bMS medium and incubated at28° C. and 200 rpm. The production of jasmonic acid in the cultures wasmonitored by TLC and HPLC analysis.

TLC Solvents Hexane 10% Ethyl acetate 90% Acetic acid 0.5% 

Microorganisms Jasmonic Acid Production (9 Days) Diplodia gossypina ATCC10936 648 ppm Diplodia gossypina ATCC 16391 — Diplodia gossypina ATCC20575 1263 ppm  Diplodia gossypina ATCC 22644 — Diplodia gossypina ATCC26123 — Diplodia gossypina ATCC 34643 — Diplodia gossypina ATCC 36037 —Diplodia gossypina ATCC 64959 — Diplodia gossypina ATCC 20571 — Diplodiagossypina NRRL 13607 — Diplodia gossypina NRRL 25011  75 ppm Diplodiagossypina ATCC 34643 503 ppm Botryodiplodia theobromae D7/2 337 ppm(German Patent Stain)

EXAMPLE III Screening of Microorganisms for the Production of JasmonicAcid in Submerged Culture Under Reduced Nitrogen Levels

Medium: Buffered Minimal Salts Medium (bMS) NaNO₃ 0.5 gm/L KH₂PO₄ 2.0gm/L K₂HPO₄ 0.5 gm/L MgSO₄-7H₂O 2.0 gm/L KCl 0.5 gm/L FeSO₄-7H₂O 1.0mg/L TASTONE ® 900 0.5 gm/L

Following sterilization of bMS medium at 120° C. and 15 psi, 60 g/l of50% sterile glucose solution was added.

Procedure

Inocula for all microorganisms were prepared from frozen cultures. 2 Mlof inoculum were added to 100 ml of sterile bMS medium and incubated at28° C. and 200 rpm. The production of jasmonic acid in the cultures wasmonitored by TLC and HPLC analysis.

TLC Solvents Hexane 10% Ethyl acetate 90% Acetic acid 0.5% 

Jasmonic Acid Production Microorganisms (7 Days) Botryodiplodia rhodinaCBS 110.1 − Botryodiplodia rhodina CBS 124.13.1 − Botryodiplodia rhodinaCBS 175.26 − Botryodiplodia rhodina CBS 176.25 44 ppm Botryodiplodiarhodina CBS 190.73 + Botryodiplodia rhodina CBS 287.47 + Botryodiplodiarhodina CBS 289.56 − Botryodiplodia rhodina CBS 304.79 + Botryodiplodiarhodina CBS 306.58 + Botryodiplodia rhodina CBS 356.59 − Botryodiplodiarhodina CBS 374.54 − Botryodiplodia rhodina CRS 447.62 + Botryodiplodiarhodina CBS 456.78 − Botryodiplodia rhodina CBS 494.78 − Botryodiplodiarhodina CBS 495.78 − Botryodiplodia rhodina CBS 559.7 + Diplodiagossypina ATCC 36037 417 ppm  Diplodia gossypina ATCC 58760 95 ppmDiplodia gossypina ATCC 76087 +

EXAMPLE IV Production of Jasmonic Acid in Stationary Fernbach FlaskCulture

Reactions:

Medium

Medium 5074H KH₂PO₄ 1.0 gm MgSO₄.7H₂O 0.5 gm Yeast extract 1.0 gm Soypeptone 5.0 gm Trace minerals 10.0 ml Dextrose 30.0 gm Deionized water1.0 L pH adjusted to 9.0 before sterilization

Trace Minerals Solution 5074H FeSO₄.7H₂O 10.0 mg ZnSO₄.7H₂O 8.8 mgCuSO₄.5H₂O 15.0 mg MnSO₄.H₂O 7.6 mg (NH₄)₆Mo₇O₂₄.4H₂O 10.0 mg Deionizedwater 1.0 L

Procedure

Inoculum:

A 500 ml Erlenmeyer flask containing 100 ml of the above medium wasinoculated with Diplodia gossypina ATCC 10936, using a stock slantculture. 10 Ml of sterile 50% dextrose were added and the flask wasincubated for 72 hours at 30° C. and 150 rpm.

Production:

500 Ml of the above medium was added to a 2.8 liter Fembach flask andsterilized for 20 minutes at 121° C. The 72 hour inoculum was chopped ina sterile blender jar, and 10 ml were added along with 50 ml of sterile50% dextrose to the Fernbach flask, followed by stationary incubation at30° C.

Results

After 10 days of incubation, 402 ml of culture broth were recovered,having a titer of 1.2 gram/liter jasmonic acid as determined by HPLC.

EXAMPLE V Production of Jasmonic Acid in Aseptic Stationary Tray Culture

Reactions:

Medium

Medium 5059H KH₂PO₄ 1.0 gm MgSO₄.7H₂O 0.5 gm Yeast extract 10.0 gm Soypeptone 5.0 gm Trace minerals 10.0 ml Dextrose 30.0 gm Deionized water1.0 L pH adjusted to 8.5 before sterilization

Trace Minerals Solution 5059H FeSO₄.7H₂O 10.0 mg ZnSO₄.7H₂O 8.8 mgCuSO₄.5H₂O 15.0 mg MnSO₄.H₂O 7.6 mg (NH₄)₆Mo₇O₂₄.4H₂O 10.0 mg Deionizedwater 1.0 L

Procedure

Inoculum:

A 500 ml Erlenmeyer flask containing 100 ml of the above medium wasinoculated with Diplodia gossypina ATCC 10936, using a stock slantculture. 10 Ml of sterile 50% dextrose were added and the flask wasincubated for 72 hours at 30° C. and 150 rpm.

Production:

3 Liters of sterile broth, adjusted to pH 8.5 before sterilization, wereinoculated with 10 ml of a 72 hour chopped inoculum. 300 Ml of sterile50% dextrose were added, the mixture poured into a sterile plastic tray(approximately 11 inches long, 10 inches wide and 5 inches deep) andcovered with a cheesecloth/cotton screen to maintain pure cultureconditions. The tray culture was incubated stationary for 14 days at31.5° C.

Results

The culture broth had a titer of 0.71 gram/liter after 10 days ofincubation. The final recovered broth (2.14 liters, 14 days) had a titerof 1.24 gram/liter as determined by HPLC.

EXAMPLE VI Tray Culture Production of Jasmonic Acid

Reactions:

Medium

Medium 5059H KH₂PO₄ 1.0 gm MgSO₄.7H₂O 0.5 gm Yeast extract 1.0 gm Soypeptone 5.0 gm Trace minerals 10.0 ml Dextrose 30.0 gm Deionized water1.0 L pH adjusted to 9 before sterilization

Chloramphenicol was added after sterilization to help protect againstcontamination.

Trace Minerals Solution 5059H FeSO₄.7H₂O 10.0 mg ZnSO₄.7H₂O 8.8 mgCuSO₄.5H₂O 15.0 mg MnSO₄.H₂O 7.6 mg (NH₄)₆Mo₇O₂₄.4H₂O 10.0 mg Deionizedwater 1.0 L

Procedure

Inoculum:

A 72 hour inoculum was prepared in 2.8 liter Fembach flasks, each flaskcontaining 100 ml. The flasks were incubated at 30° C. and 150 rpm.

Production:

A total of 540 liters of broth was prepared in a 300 gallon fermenter.The pH was adjusted to 9.0, and the broth was sterilized for 30 minutesat 121° C. After cdoling to 30° C., chloramphenicol (36 grams dissolvedin 600 ml ethanol), 60 liters of sterile 50% dextrose and 2,307 gramschopped inoculum (weight adjusted for a standard 45% packed cell volume)were added. After mixing for 5-10 minutes, the inoculated broth wastransferred to large plastic trays (approximately 20 inches long, 17inches wide and 5 inches deep), placed on shelves in anenvironmentally-controlled room. Approximately 6 liters of inoculatedbroth was added to each tray (total of 100 trays), the trays coveredwith aluminum foil and incubated stationary at 31-32° C. for 14 days. Atthe conclusion of incubation, the mycelial mats were filtered off, thebroth was transferred to the 300 gallon fermenter and sterilized for 20minutes at 121° C. The broth was cooled to room temperature, acidifiedto pH 4, and the jasmonic acid was recovered by adsorption/elution on anoniomc resm.

Results

The bulked broth (approximately 380 liters) from the 100 tray cultureshad a titer of 0.895 gram/liter jasmonic acid as determined by HPLC.

EXAMPLE VII Production of Jasmonic Acid by Combined Surface-SubmergedFermentation

Reactions:

Medium

Medium 5059H KH₂PO₄ 1.0 gm MgSO₄.7H₂O 0.5 gm Yeast extract 1.0 gm Soypeptone 5.0 gm Trace minerals 10.0 ml Dextrose 30.0 gm Deionized water1.0 L pH adjusted to 9 before sterilization

Chloramphenicol was added after sterilization to help protect againstcontamination.

Trace Minerals Solution 5059H FeSO₄.7H₂O 10.0 mg ZnSO₄.7H₂O 8.8 mgCuSO₄.5H₂O 15.0 mg MnSO₄.H₂O 7.6 mg (NH₄)₆Mo₇O₂₄.4H₂O 10.0 mg Deionizedwater 1.0 L

Procedure

Inoculum:

A 500 ml Erlenmeyer flask containing 100 ml of the above medium wasinoculated with Diplodia gossypina ATCC 10936, using a stock slantculture. 10 Ml of sterile 50% dextrose were added and the flask wasincubated for 72 hours at 30° C. and 150 rpm.

Production:

A single coil of stainless steel wire cloth, having approximately ⅜inch×⅜ inch openings, was attached vertically to the baffles of a 150liter fermenter. Four circular wire cloth screens (same opening size)were attached to the vertical coil in horizontal positions such that thelowest screen was about one inch above the broth surface, and the otherscreens were separated by about one inch space between each screen. Anopening in the center of each screen permitted freedom for agitation,using one impeller.

60 Liters of broth were made up and transferred to the fermenter, the pHadjusted to 9.0 and the broth sterilized at 121° C. for 30 minutes.After cooling to 30° C., 2.4 liters of chopped 72 hour inoculum,chloramphenicol (6.0 grams dissolved in 60 ml of ethanol) and 1.2 litersof sterile 50% dextrose were added. Sterile 50% dextrose solution andsterile deionized water were added as needed during the 19 dayincubation at 30° C. Aeration was provided and agitation was engagedperiodically to prevent the developing mycelium attached to the screensfrom drying out.

Results

At the conclusion of the incubation, approximately 31 liters of brothwere recovered, having a titer of 185 mg/liter jasmonic acid asdetermined by HPLC.

EXAMPLE VIII Production of Jasmonic Acid Using Plastic BiofilterSupports

Reactions:

Medium

Medium 5059H KH₂PO₄ 1.0 gm MgSO₄.7H₂O 0.5 gm Yeast extract 1.0 gm Soypeptone 5.0 gm Trace minerals 10.0 ml Dextrose 30.0 gm Deionized water1.0 L pH adjusted to 9 before sterilization

Chloramphenicol was added after sterilization to help protect againstcontamination.

Trace Minerals Solution 5059H FeSO₄.7H₂O 10.0 mg ZnSO₄.7H₂O 8.8 mgCuSO₄.5H₂O 15.0 mg MnSO₄.H₂O 7.6 mg (NH₄)₆Mo₇O₂₄.4H₂O 10.0 mg Deionizedwater 1.0 L

Procedure

Inoculum:

A 500 ml Erlenmeyer flask containing 100 ml of the above medium wasinoculated with Diplodia gossypina ATCC 10936, using a stock slantculture. 10 Ml of sterile 50% dextrose were added and the flask wasincubated for 72 hours at 30° C. and 150 rpm. The pH of the broth wasadjusted to 9.0 before sterilization.

Production:

A 130 liter fermenter, equipped with a spray ball attached to the centerof the headplate, was filled ⅔ full with Jaeger Tripack biofilter 2 inchdiameter supports. 30 Liters of broth was prepared and, after the pH wasadjusted to 9.0, was charged to the fermenter. A recirculation loop wasprovided for the purposes of broth sterilization, for periodic brothrecirculation (through the spray nozzle) during incubation to replenishnutrients to the developing mycelium attached to the biosupports and toprevent drying out. Sterilization was carried out by continuousrecirculation for 30 minutes at 121° C. After cooling to 30° C., 30 mlof chopped inoculum, chloramphenical (3 grams dissolved in 30 ml ofethanol) and 3 liters of sterile 50% dextrose were added. Incubation wascarried out at 30° C. without agitation, and periodic recirculation ofthe inoculated broth was performed. Aeration was provided, and sterile50% dextrose solution and deionized water were added as needed.

Results

Based on the original 30 liters of starting broth, the batch yielded 146mg/liter jasmonic acid as determined by HPLC.

EXAMPLE IX Production of Jasmonic Acid in Shake Flask Culture

Reactions:

Medium: Buffered Minimal Salts Medium (bMS) NaNO₃ 0.5 gm/L KH₂PO₄ 2.0gm/L K₂HPO₄ 0.5 gm/L MgSO₄-7H₂O 2.0 gm/L KCl 0.5 gm/L FeSO₄-7H₂O 1.0mg/L TASTONE ® 900 0.5 gm/L

Following sterilization of bMS medium at 120° C. and 15 psi, 60 g/l of50% sterile glucose solution was added.

Procedure

Inoculum:

A 500 ml Erlenmeyer flask containing 100 ml of sterile bMS medium wasinoculated with 2 ml of a frozen chopped cell culture of Diplodiagossypina ATCC 10936. 6 Ml of sterile 50% dextrose were added and theflask was incubated for 3 days at 28° C. and 200 rpm. Following 3 daysof incubation, the culture was chopped for 1 minute in a sterile WaringBlender and this was used as inoculum.

Production:

1 Ml of inoculum was added to 100 ml of sterile bMS medium in a 500 mlErlenmeyer flask. 6 Ml of sterile 50% dextrose were added and the flaskwas incubated at 28° C. and 200 rpm. The production of jasmonic acid inthe culture was monitored by TLC and HPLC analyses.

Results

Diplodia gossypina ATCC 10936 produced 1.2 gram/liter jasmonic acidafter 7 days of incubation.

EXAMPLE X Production of Jasmonic Acid in Fernbach Flask SubmergedCulture

Reactions:

Medium: Buffered Minimal Salts Medium (bMS) NaNO₃ 2.0 gm/L KH₂PO₄ 2.0gm/L K₂HPO₄ 0.5 gm/L MgSO₄-7H₂O 2.0 gm/L KCl 0.5 gm/L FeSO₄-7H₂O 1.0mg/L TASTONE ® 900 0.5 gm/L

After sterilization of bMS medium at 120° C. and 15 psi 60 g/l of 50%sterile glucose solution was added;

Procedure

Inoculum:

A 500 ml Erlenmeyer flask containing 100 ml of sterile bMS medium wasinoculated with 2 ml of a frozen chopped cell culture of Diplodiagossypina ATCC 10936. 6 Ml of sterile 50% dextrose were added and theflask was incubated for 3 days at 28° C. and 200 rpm. Following 3 day ofincubation, the culture was chopped for 1 minute in a sterile WaringBlender and this was used as inoculum.

Production:

5 Ml of inoculum were added to 500 ml of sterile bMS medium in a Fembachflask. 30 Ml of sterile 50% dextrose were added and the flask wasincubated at 28° C. and 150 rpm. The production ofjasmonic acid wasmonitored by TLC and HPLC analyses.

Results

Diplodia gossypina ATCC 10936 produced jasmonic acid at a titer of 0.9gram/liter after 8 days of incubation.

EXAMPLE XI Production of Jasmonic Acid in Laboratory Fermenters

Reactions:

Medium: Buffered Minimal Salts Medium (bMS) NaNO₃ 1.0 gm/L KH₂PO₄ 2.0gm/L K₂HPO₄ 0.5 gm/L MgSO₄-7H₂O 2.0 gm/L KCl 0.5 gm/L FeSO₄-7H₂O 1.0mg/L TASTONE ® 900 0.5 gm/L

Following sterilization of bMS medium at 120° C. and 15 psi, 60 g/1 of50% sterile glucose solution was added.

Procedure

Inoculum:

A 500 ml Erlenmeyer flask containing 100 ml of sterile bMS medium wasinoculated with 2 ml of a frozen chopped cell culture of Diplodiagossypina ATCC 10936. 6 Ml of sterile 50% dextrose were added and theflask was incubated for 3 days at 28° C. and 200 rpm. Following 3 daysof incubation, the inoculum was chopped for 1 minute in a sterile WaringBlender. 1 M1 of inoculum and 6 ml of sterile 50% dextrose were added to100 ml of sterile bMS medium. After another 3 days of incubation, thewhole culture was used as an inoculum for a laboratory 10 literfermenter.

Production:

10 Liters of bMS medium containing 1 gram/liter of NaNO₃ was added to a14 liter laboratory fermenter and sterilized for 30 minutes at 121° C.The above inoculum and 600 grams of sterile 50% dextrose were added to afermenter. The fermenter was run at 28° C. and 1,000 rpm with aerationat 1 VVM and 15 psi back pressure. The pH of fermenter was maintained at6.0 by addition of 25% NaOH. After 4 days of incubation, an additional0.5 gram/liter aliquot of NaNO₃ was added. The concentration of glucosein the broth was monitored and additional sterile 50% dextrose was addedto prevent glucose depletion. The production of jasmonic acid in thebroth was monitored by TLC and HPLC analyses.

Results

After 13 days of incubation, Diplodia gossypina ATCC 10936 producedjasmonic acid at a titer of 0.85 gram/liter.

EXAMPLE XII Production of Jasmonic Acid in Submerged 100-L Pilot PlantFermenters

Reactions:

Medium: Buffered Minimal Salts Medium (bMS) NaNO₃ 0.5 gm/L KH₂PO₄ 2.0gm/L K₂HPO₄ 0.5 gm/L MgSO₄-7H₂O 2.0 gm/L KCl 0.5 gm/L FeSO₄-7H₂O 1.0mg/L TASTONE ® 900 0.5 gm/L

Following steriliation of bMS medium at 120° C. and 15 psi, 60 g/l of50% sterile glucose solution was added.

Procedure

Inoculum:

A 500 ml Erlenmeyer flask containing 100 ml of sterile bMS medium wasinoculated with 2 ml of a frozen chopped cell culture of Diplodiagossypina ATCC 10936. 6 Ml of sterile 50% dextrose were added and theflask was incubated for 3 days at 28° C. and 200 rpm. Following 3 daysof incubation, the inoculum was chopped for 1 minute in a sterile WaringBlender. 5 Ml of the inoculum and 30 ml of sterile 50% dextrose wereadded to each of two Fembach flasks containing 500 ml of sterile bMSmedium. The flasks were incubated at 28° C. and 150 rpm for 72 hours.

Production:

100 Liters of bMS containing 1 gram/liter of NaNO₃ were charged to a 150liter pilot plant fermenter and sterilized for 30 minutes at 121° C.1,000 Ml of whole inoculum and 600 grams of sterile 50% dextrose wereadded to the fermenter. The fermenter was run at 28° C. and 450 rpm withaeration at 1 VVM and 15 psi back pressure. The pH of fermenter wasmaintained at 6.0 by addition of 25% NaOH. After 4 days of incubation,an additional 0.5 gram/liter aliquot of NaNO₃ was added. Theconcentration of glucose in the broth was monitored and additionalsterile 50% dextrose was added to prevent glucose depletion. Theproduction of jasmonic acid in cell culture was monitored by TLC andHPLC analyses.

Results

After 9 days of incubation, Diplodia gossypina ATCC 10936 producedjasmonic acid at a titer of 0.85 gram/liter.

EXAMPLE XIII Effect of 10-oxo-8-trans-decenoic Acid on the Production ofJasmonic Acid in Fernbach Flask Submerged Culture

Reactions:

Medium: Buffered Minimal Salts Medium (bMS) NaNO₃ 0.5 gm/L KH₂PO₄ 2.0gm/L K₂HPO₄ 0.5 gm/L MgSO₄-7H₂O 2.0 gm/L KCl 0.5 gm/L FeSO₄-7H₂O 1.0mg/L TASTONE ® 900 0.5 gm/L

Following sterilization of bMS medium at 120° C. and 15 psi, 60 g/l of50% sterile glucose solution was added.

Procedure

Inoculum:

A 500 ml Erlenmeyer flask containing 100 ml of sterile bMS medium wasinoculated with 2 ml of a frozen chopped cell culture of Diplodiagossypina ATCC 10936. 6 Ml of sterile 50% dextrose were added and theflask was incubated for 3 days at 28° C. and 200 rpm. Following 3 daysof incubation, the culture was chopped for 1 minute in a sterile WaringBlender and this was used as inoculum.

Production:

5 Ml of inoculum and 30 ml of sterile 50% dextrose were added to aFernbach flask containing 500 ml of sterilize bMS medium. A stocksolution of 50% 10-oxo-decenoic acid (10-ODA) in ethanol was preparedand dispensed to the flasks at a final concentration of either 1 or 10ppm. The cultures were incubated at 28° C. and 150 rpm. The productionofjasmonic acid was monitored by TLC and HPLC analyses.

Results

Number Flasks 3 Days 6 Days 1 Control 106 ppm 249 ppm 2  1 ppm ODA 197ppm 538 ppm 3 10 ppm ODA 279 ppm 630 ppm

EXAMPLE XIV Effect of 10-oxo-8-trans-decenoic Acid on the Production ofJasmonic Acid in a Submerged 10-L of Laboratory Fermenter

Reactions:

Medium: Buffered Minimal Salts Medium (bMS) NaNO₃ 2.0 gm/L KH₂PO₄ 2.0gm/L K₂HPO₄ 0.5 gm/L MgSO₄-7H₂O 2.0 gm/L KCl 0.5 gm/L FeSO₄-7H₂O 1.0mg/L TASTONE ® 900 0.5 gm/L

After sterilization of bMS medium at 120° C. and 15 psi, 60 g/l of 50%sterile glucose solution was added.

Procedure

Inoculum:

A 500 ml Erlenmeyer flask containing 100 ml of sterile bMS medium wasinoculated with 2 ml of a frozen chopped cell culture of Diplodiagossypina ATCC 10936. 6 Ml of sterile 50% dextrose were added and theflask was incubated for 3 days at 28° C. and 200 rpm. Following 3 daysof incubation, the inoculum was chopped for 1 minute in a sterile WaringBlender. 1 Ml of inoculum and 6 ml of sterile 50% dextrose were added to100 ml of sterile bMS medium. After another 3 days of incubation, thewhole culture was used as an inoculum for a laboratory 10 literfermenter.

Production:

10 Liters of bMS containing 1.0 grams/liter of NaNO₃ were charged to a14 liter laboratory fermenter and sterilized for 30 minutes at 121° C.100 Ml of inoculum lated and 600 grams of sterile 50% dextrose wereadded to the fermenter. A stock solution of 10-ODA was prepared as inExample XIII and added to the fermenter at a final concentration of 10ppm in the broth. The fermenter was run at 28° C. and 1,000 rpm withaeration at 1 VVM and 15 psi back pressure. The pH of fermenter wasmaintained at 6.0 by the addition of 25% NaOH. After 4 days ofincubation, an additional 0.5 grams/liter aliquot of NaNO₃ was added.The concentration of glucose in the broth was monitored and additionalsterile 50% dextrose was added to prevent glucose depletion. Theproduction ofjasmonic acid was monitored by TLC and HPLC analyses.

Results

After 11 days of incubation, Diplodia gossypina ATCC 10936 producedjasmonic acid at a titer of 1.5 grams/liter.

EXAMPLE XV Production of Natural “EPI” Methyl Jasmonate-Preparation andPurification from Natural Jasmonic Acid Fermentation Broth Extract

Reactions:

Description of Process Steps

1.0 The rush-over of the crude topped extract is carried out under bestvacuum to a final pot temperature of 260C. At higher temperatures, thereis thermal breakdown and some loss of vacuum. Most of the charge remainsas “residue.” A portion of this residue was dissolved in ethyl acetateand extracted with aqueous base. Re-acidification and GLC analysisindicated that there was no jasmonic acid left remaning in the residue.

2.0. The extract is stirred and 3 liters of 6% sodium carbonate solutionto neutralize the sodium jasmonate. The pH is adjusted to 9.

3.0. The basic solution is extracted three times with hexane. Thisremoves non-acidic impurities as well as significant sulfurous odorbodies. At lower pH, more material is removed.

The hexane extract contains approximately 160 grams of organic material.The major comonent is identified as a lactone having the structure:

This lactone is derived from the alcohol according to the reaction:

4.0. Approximately 225 grams of 85% phosphoric acid is required toadjust the acidity to pH=2.

5.0 N asm onic acid having the structures:

 is charged to an autoclave along with 4 times its weight of naturalmethanol. The autoclave is sealed and operated for a period of 12 hoursat 150° C. The final composition is approximately 70:30 (weight:weight)methyl jasmonate:jasmonic acid. The autoclave pressure is 220 psig.

6.0 The autoclave is opened and the methanol stripped from the resultingproduct.

The pH of the product is adjusted to 9.

7.0 Ethyl acetate is used to extract the resulting methyl jasmonate. Theethyl acetate is stripped at a temperature below 60° C. at atmosphericpressure. The resulting methyl jasmonate is then fractionally distilled,using a 12 inch×1.5 inch Goodloe packed column. The first fraction isdistilled at a reflux ratio of 4:1, and the remainder of the fractionsare distilled at reflux ratio of 1:1. The conditions of distillationare: 125° C.; vapor temperature at 1 mm/Hg pressure.

8.0 The methyl jasmonate is redistilled at a reflux ratio of 4:1 at 125°C. and 1 mm/Hg pressure.

EXAMPLE XVI

A white chocolate raspberry flavor was prepared for addition to nonfatyogurt containing aspartame.

The following flavor composition was prepared:

Ingredients Parts by Weight Jasmonic acid prepared according to ExampleVI 15 Natural cocoa extract 28 Natural raspberry extract 303-Phenyl-4-pentenal  8

At the rate of 8 ppm, the above-mentioned mixture was added to naturalnonfat yogurt containing aspartame, cultured, pasturized, grade A nonfatmilk, modified cornstarch, whey protein and gelatin, and active cultureswith L. acidophilus.

On mixing the flavor with the natural yogurt, the resulting product hasa natural raspberry, fresh, “just-picked,” “seedy” taste with floraltopnote.

A second sample was flavored without the jasmonic acid present, and theresulting flavor did not have the “fresh-just picked-seedy” quality.

EXAMPLE XVII Jasmine Perfume Formulation

The following mixture is prepared:

Parts by Ingredients Weight Jasmonic acid prepared according to ExampleXIV 50 Methyl jasmonate product prepared according to Example 50 XVOrange oil 20 Bergamot oil 20 Neroli oil 20 γ-Methyl ionone 201-Acetyl-2,5,5-trimethyl cycloheptane 45

The products of Examples XIV and XV impart to this jasmine perfumeformulation powerful, long lasting jasmine, floral-herbaceous aromaswith sweet-herbaceous, green-woody topnotes. Accordingly, the perfumecomposition of Example XVII can be described as “jasmine withfloral-herbaceous undertones and sweet-herbaceous, green-woodytopnotes.”

EXAMPLE XVIII Preparation of Soap Compositions

100 Grams of soap chips are produced according to Example V of U.S. Pat.No. 4,058,487 issued on Nov. 5, 1997, the specification for which isincorporated herein by reference, as follows:

The sodium salt of an equal mixture of C₁₀-C₁₄ alkane sulfonate (95%active), 40 lbs, is dissolved in a mixture of 80 lbs of anhydrousisopropanol and 125 lbs of deionized water at 150° F. In this mixture isdissolved 10 lbs of partially hydrogenated coconut oil fatty acids and15 lbs of sodium mono-C₁₄ alkyl maleate, and the pH of this solution isadjusted to 6.0 by the addition of a small amount of 50% aqueoussolution of sodium hydroxide. The isopropanol is distilled off and theremaining aqueous solution is drum dried. The resulting solid activesare then blended in a chip mixture with 10 lbs of water, 0.2 lbs oftitanium hydroxide and 0.7 lbs of one of the perfume ingredients setforth in Table V, infra.

TABLE V Ingredients Fragrance Profile Jasmonic acid isomer having the Apowerful, long lasting jasmine, structure: floral-herbaceous aroma withsweet- herbaceous, green-woody topnotes.

(optical rotation: +58°) Isomer having the structure: A powerful, longlasting jasmine, floral-herbaceous aroma with sweet- herbaceous,green-woody topnotes.

(optical rotation: +58°). Isomer having the structure: A powerful, longlasting jasmine, floral-herbaceous aroma with sweet- herbaceous,green-woody topnotes.

(optical rotation: +58°). Isomer having the structure: A powerful, longlasting jasmine, floral-herbaceous aroma with sweet- herbaceous,green-woody topnotes.

(optical rotation: +58°). Perfume composition of Jasmine with floral,herbaceous Example XVII. undertones and sweet, herbaceous, green, woodytopnotes.

EXAMPLE XIX Preparation of Detergent Composition

A total of 100 grams of a detergent powder prepared according to U.S.Pat. No. 4,058,472 (the specification for which is incorporated byreference herein) and containing 5% by weight of the sodium salts of amixture of sulfonated C₁₄-C₁₈ alkyl catechol as a surface activecomponent, the mixture being 60 parts by weight of mono-C₁₄-C₁₈ alkylcatechol and 40 parts by weight of di-C₁₄-C₁₈ catechol, 35% sodiumtetrapyrophosphate, 30% sodium silicate, 20% of sodium carbonate, 3% ofsodium carboxymethyl cellulose and 7% of starch is mixed with 0.15 gramsindividually with each of the aroma ingredients set forth in Table V ofExample XVIII until a substantially homogeneous composition is obtained.Each of the compositions has an excellent aroma as set forth in Table Vof Example XVIII.

EXAMPLE XX Preparation of a Cosmetic Powder Composition

A cosmetic powder is prepared by mixing in a ball mill, 100 grams oftalcum powder with 0.25 grams of each of the perfume materials of TableV of Example XVIII. Each of the powders has an excellent aroma as setforth in Table V of Example XVIII.

EXAMPLE XXI Perfumed Liquid Detergent

Concentrated liquid detergents with aromas as set forth in Table V ofExample XVIII are prepared by adding 0.10%, 0.15% and 0.20% of each ofthe ingredients set forth in Table V of Example XVIII. They are preparedby adding and homogeneously mixing the appropriate quantity of perfumesubstance of Table V of Example XVIII in the liquid detergent. Thedetergents individually possess aromas as set forth in Table V ofExample XVIII, the intensity increasing with greater concentrations ofperfume substance set forth in Table V of Example XVIII.

EXAMPLE XXII Preparation of a Cologne and Handkerchief Perfume

Each of the ingredients of Table V of Example XVIII is incorporatedindividually into colognes of several strengths at concentrations of2.0%, 2.5%, 3.0%, 3.5%, 4.0% and 5.0% in 75%, 80%, 85%, 90% and 95%aqueous ethanol; and into several concentrations of handkerchiefperfumes at the rate of 15%, 20% and 25% (in 80%, 85%, 90% and 95%aqueous ethanol). Distinct and defnite aromas as set forth in Table V ofExample XVIII are imparted to the colognes and to the handkerchiefperfimes at the several concentrations set forth above.

EXAMPLE XXIII Preparation of Soap Compositions

100 Grams of soap chips (IVORY®, produced by the Procter & GambleCompany of Cincinnati, Ohio) are admixed with 1 gram of each of thesubstances set forth in Table V of Example XVIII, supra, untilhomogeneous compositions are heated under 3 atmospheres pressure at 180°C. for a period of 3 hours, and the resulting liquids are placed intosoap molds. The resulting soap cakes, on cooling, manifest excellentaromas as set forth in Table V of Example XVIII.

EXAMPLE XXIV Preparation of Solid Detergent Compositions

Detergents are prepared from the following ingredients according toExample I of Canadian Patent No. 1,007,948, the specification for whichis incorporated by reference herein:

Parts Ingredients by Weight NEODOL ® 45-11 (a C₁₄-C₁₅ alcoholethoxylated with 12 11 moles of ethylene oxide) Sodium carbonate 55Sodium citrate 20 Sodium sulfate, water brighteners q.s.

This detergent is a “phosphate-free” detergent. A total of 100 grams ofsaid detergent is admixed with 0.10, 0.15, 0.20 and 0.25 grams of eachof the substances set forth in Table V of Example XVIII, supra. Each ofthe detergent samples has an excellent aroma as indicated in Table V ofExample XVIII.

EXAMPLE XXV Preparation of Drier-Added Fabric Softener Article

Utilizing the procedure of Example I at column 15 of U.S. Pat. No.3,632,396, the specification for which is incorporated by referenceherein, a non-woven cloth substrate useful as a drier-added fabricsoftening article of manufacture is prepared wherein the substrate,substrate coating, outer coating and the perfume material are asfollows:

1. a water “dissolvable” paper (“Dissolvo Paper”) as the substrate;

2. ADOGEN® 448 (melting point about 140° F.) as the first substratecoating; and

3. an outer coating having the following formulation (melting pointabout 150° F.):

57% C₂₀-C₂₂ HAPS;

22% isopropyl alcohol;

20% antistatic agent; and

1% of one of the perfumery substances set forth in Table V of ExampleXVIII, supra.

Fabric softening compositions containing the substances as set forth inTable V of Example XVIII, supra, essentially consist of a substratehaving a weight of about 3 grams per 100 square inches; a substratecoating weighing about 1.85 grams per 100 square inches of substrate;and an outer coating weighing about, 1.5 grams per 100 square inches ofsubstrate are prepared, thereby providing a total aromatized substrateand outer coating weight ratio of about 1:1 by weight of the substrate.

The aromas as set forth in Table V of Example XVIII supra, are impartedin a pleasant manner to the headspace in a drier on operation thereof,using the said drier-added fabric softening non-woven fabric by addingto the drying cycle.

As stated above in the case of fabric softener articles, the entire U.S.Pat. No. 3,632,396 is incorporated by reference herein. Thus, all of thearticles of U.S. Pat. No. 3,632,396, acting as fabric softening articlesin said U.S. Pat. No. 3,632,396, may be perfumed in their outer coatingwith from 0.25% up to 5% by weight of each of the perfiuming substancesof Table V of Example XVIII, supra.

EXAMPLE XXVI Hair Preparation

A “soft-feel, good-hold” hair spray is produced containing the followingingredients:

Parts by Ingredients Weight Polyvinylpyrollidone/vinyl acetate “E-735Copolymer” 4.00 manufactured by the GAF Corporation of New York, NYAnhydrous ethanol 70.90 Dioctyl sebecate 0.05 Benzyl alcohol 0.05“Propellant A-46” manufactured by the GAF Corporation of 24.95 New York,NY Fragrance ingredient as set forth in Table V of Example XVIII, 0.05supra

The PVP/VA copolymers are first dissolved in alcohol and all otheringredients are added until uniform. The propellant is then pressurizedand used as an aerosol. The resulting hair sprays each have pleasantaromas as set forth in Table V of Example XVIII, supra.

EXAMPLE XXVII Scouring Cleanser Composition

A scouring cleanser composition is prepared in accordance with Example Iat columns 11 and 12 of U.S. Pat. No. 4,193,888 issued on Mar. 18, 1980,the specification for which is incorporated by reference herein. To thiscomposition, the substances set forth in Table V of Example XVIII,supra, are added at the level of 0.25% as set forth in the table in saidExample I of U.S. Pat. No. 4,193,888, yielding an aroma on using saidcleanser in ordinary circumstances which is quite pleasant and describedin Table V of Example XVIII, supra.

EXAMPLE XXVIII

A fabric softening article prepared substantially as set forth inExample VIII of Canadian Patent No. 1,069,260, the specification forwhich is incorporated by reference herein, is prepared containing 0.21%by weight of a perfuming substance as set forth in Table V of ExampleXVIII supra, and yielding, on use in a drier, a faint aroma as set forthin Table V of Example XVIII, supra.

EXAMPLE XXIX Tobacco Flavor Formulations

Cigarettes are produced using the following formulations:

Ingredients Parts by Weight Bright 40.1 Burley 24.9 Maryland 1.1 Turkish11.6 Stem (flue cured) 14.2 Glycerine 2.8 H₂O 5.3

At the rate of 0.2%, the following tobacco formulation is applied to allof the cigarettes produced with the above tobacco formulation:

Parts by Ingredients Weight Ethyl butyrate 50 Ethyl valerate 50 Maltol20 Cocoa extract 20 Coffee extract 20 Ethyl alcohol (95%) 45 H₂O 41.900

To portions of 50% of the cigarettes at levels of 10 and 20 ppm, thejasmonic acid-containing composition of Example VI is added. Thesecigarettes are hereinafter called “experimental” cigarettes. Thecigarettes without the jasmonic acid composition are hereinafter called“control” cigarettes. The control and experimental cigarettes are thenevaluated by paired comparison and the results are as follows:

(a) in aroma, the experimental cigarettes are all found to be morearomatic with Turkish tobacco-like nuances; and

(b) in smoke flavor, the experimental cigarettes are all found to bemore aromatic, more sweet with Turkish tobacco, oriental-like nuancesthan the control cigarettes.

The experimental cigarettes containing the mixture of jasmonic acids arefound to be fruity and floral and have pleasant, aesthetically pleasingfruity and floral notes, in addition.

What is claimed is:
 1. A process for the manufacture of food flavoracceptable jasmonic acid in relatively high yield of from 1-2 grams perliter, containing at least 5% of a “cis” isomer having a structureselected from the group consisting of:

and a maximum of 95% by weight of at least one of the isomers having astructure selected from the group consisting of:

which comprises the steps of (i) cultivation under aerobic conditions ofa strain of Diplodia gossypina organism selected from the groupconsisting of: (a) Diplodia gossypina ATCC 10936; (b) Diplodia gossypinaATCC 20575; (c) Diplodia gossypina NRRL 25011; and (d) Diplodiagossypina ATCC 36037 in a nutrient medium containing an assimilablesource of carbon and an assimilable source of nitrogen followed by (ii)isolation of the jasmonic acid product from the culture medium.
 2. Theprocess of claim 1 wherein the compound 10-oxo-trans-8-decenoic acidhaving the structure:

is intimately admixed in the nutrient medium in an amount of from about0.7 up to about 10 ppm by weight of the nutrient medium prior to thecultivation step.
 3. The process of claim 1 wherein the cultivationtakes place under aeration conditions at an aeration rate in the rangeof from about 0.5 V/V/M up to about 1.0 V/V/M.
 4. The process of claim 2wherein the cultivation takes place under aeration conditions at anaeration rate in the range of from about 0.5 V/V/M up to about 1.0V/V/M.
 5. The process of claim 1 wherein the nutrient medium containsfrom about 6×10⁻³ up to about 10.5×10⁻³ gram moles/liter of [Mg⁺⁺]. 6.The process of claim 5 wherein the source of the [Mg⁺⁺] is MgSO₄.7H₂Oand the range of concentrations of said MgSO₄.7H₂O is from about 1.5 upto about 2.5 grams per liter.
 7. The process of claim 1 wherein thestrain of Diplodia gossypina is Diplodia gossypina ATCC
 10936. 8. Theprocess of claim 7 wherein the compound 10-oxo-trans-8-decenoic acidhaving the structure:

is intimately admixed in the nutrient medium in an amount of from about0.7 up to about 10 ppm by weight of the nutrient medium prior to thecultivation step.
 9. The process of claim 7 wherein the cultivationtakes place under aeration conditions at an aeration rate in the rangeof from about 0.5 V/V/M up to about 1.0 V/V/M.
 10. The process of claim7 wherein the nutrient medium contains from about 6×10⁻³ up to about10.5×10⁻³ gram moles/liter of [Mg⁺⁺].
 11. The process of claim 2 whereinthe nutrient medium contains from about 6×10⁻³ up to about 10.5×10⁻³gram moles/liter of [Mg⁺⁺].
 12. The process of claim 1 wherein methyljasmonate containing at least 25% by weight of the isomer having thestructure:

is produced from the jasmonic acid by means of reaction of the resultingjasmonic acid with methyl alcohol according to the reaction:

wherein the final isolation comprises the steps of: (a) extraction ofthe jasmonic acid from the fermentation broth with an extraction solventto form a jasmonic acid extract; (b) concentration of the resultingjasmonic acid extract, whereby the extraction solvent is stripped; (c)esterification of the resulting jasmonic acid with methyl alcohol,whereby methyl jasmonate is formed according to the reaction:

(d) concentration of the resulting methyl jasmonate.
 13. The process ofclaim 1 wherein the jasmonic acid produced is at least 98% of theoptical isomer having the structure:

having an optical rotation (α_(D) ²⁰) of +58° and comprising theadditional steps of (iii) extraction of the resulting jasmonic acidproduct with a solvent in order to form a jasmonic acid product solventextract, (iv) concentrating the resulting extract by means of solventevaporation, thereby forming a concentrate; (v) fractionating theconcentrate with silica gel in order to effect isomer separation; and(vi) collecting said 98% optical cis isomer.
 14. The process of claim 1carried out in liquid surface culture.
 15. The process of claim 1carried out in submerged liquid culture.
 16. The process of claim 15wherein the cultivation takes place under aeration conditions at anaeration rate in the range of from about 0.5 V/V/M up to about 1.0V/V/M.
 17. The process of claim 1 wherein the assimilable carbon sourceis glucose.
 18. The process of claim 1 wherein the assimilable nitrogensource is sodium nitrate.
 19. The process of claim 1 wherein theincubation time is from about 3 up to about 15 days.
 20. The process ofclaim 1 wherein the pH range is from about 4.5 up to about
 9. 21. Theprocess of claim 20 wherein the pH of the fermentation broth is
 6. 22.The process of claim 1 wherein the temperature range of reaction is fromabout 20° C. up to about 35° C.
 23. The process of claim 1 wherein thetemperature range of the fermentation is from about 25° C. up to about32° C.